CN104479036A - Preparation method of sulfated gynostemma pentaphylla polysaccharide - Google Patents
Preparation method of sulfated gynostemma pentaphylla polysaccharide Download PDFInfo
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Abstract
The invention discloses a preparation method of sulfated gynostemma pentaphylla polysaccharide in order to solve the technical problem that the sulfated gynostemma pentaphylla polysaccharide prepared by an existing method is low in immunological competence. The preparation method comprises the following steps: adding sulfur trioxide-pyridine, an organic solvent N and N-dimethyl formamide into a three-mouth flask, completely dissolving and then adding gynostemma pentaphylla polysaccharide powder; arranging the system in a preheated water-bath device to have a constant-temperature reaction; afterwards cooling the system to room temperature; regulating the pH to 7.0 by using a NaOH liquid; adding ethanol for precipitation for one night; and centrifuging and drying in the second day to obtain the sulfated gynostemma pentaphylla polysaccharide. The preparation method disclosed by the invention is used for preparing a gynostemma pentaphylla polysaccharide product by adopting a three-factor three-level process and measuring the degree of substitution of the gynostemma pentaphylla polysaccharide product by adopting a barium chloride-gelatin turbidimetric method. The prepared sulfated gynostemma pentaphylla polysaccharide product is relatively high in immunological competence.
Description
Technical field
The present invention relates to a kind of preparation method of gynostemma pentaphylla polysaccharide, particularly relate to a kind of preparation method of sulphating gynostemma pentaphylla polysaccharide.
Background technology
Gynostemma pentaphylla is also known as paradise grass, Chao Rencan, Herb Gynostemmae Pentaphylli, Herba Gynostemmatis, Rhizome or herb of Fiveleaf Gynostemma and gynostemma pentaphyllum makino etc., and being Shaanxi Characteristic natural resource, is Curcurbitaceae gynostemma pentaphyllum genus herbaceous perennial vine plant, belongs to the plant of integration of drinking and medicinal herbs.Cold in nature, taste is sweet, useful gas, calms the nerves, hypotensive effect, and " elixir grass " being called " mystery " among the people, is classified as in Spark Program first of " rare traditional Chinese medicine ".Gynostemma pentaphylla principle active component is polyose, flavonoid, saponins and microelement kind, its effect mainly promotes body fat class substance metabolism, nutrition human body cell, there is good detoxicating, relieving inflammation effect simultaneously, be widely used in hyperlipidemia, fatty liver, obesity, constipation, insomnia, hepatitis B, the treatment of chronic airway inflammation (pharyngolaryngitis, gastroenteritis etc.) and health care.
The compound that polysaccharide is made up of with α-or β-glycosidic link much identical or different monose, is prevalent in natural plant body, comprises starch, Mierocrystalline cellulose, saccharan, pectin etc.Think that polysaccharide is most important chemical active ingredient in gynostemma pentaphylla plant at present, there is the biological activitys such as immunity moderation function, Tumor suppression, anti-oxidant, hypoglycemic, antifatigue, protection liver injury, the damage of neuroprotective cell glucose oxidation.Be widely used in makeup, food service industry.
Molecular modification has very important impact to polysaccharide biological activity, former active polysaccharide can be made to improve, or produce new activity by molecular modification.Modification means the most frequently used at present mainly adopt the method for chemistry to introduce other group, change the structure of polysaccharide.Namely sulphation modification is a kind of modifying method the most important and common during polysaccharide molecule is modified.Sulphation modification is replaced the C-terminal of polysaccharide molecule, C-terminal or N-terminal sulfate group, to obtain the sulfated polysaccharides molecule that activity is better than former polysaccharide molecule.Molecular weight and monosaccharide residue sulfate radical substitution value are most important 2 indexs.Many polysaccharide are after sulphation modification, and its immunocompetence significantly strengthens, and are mainly reflected in splenic lymphocyte and the aspect such as the propagation of scavenger cell, the secretion of stimulating cytokine, and its immunocompetence size is by the impact of substitution value.Sulphation modification can potentially as improving one of immunocompetent effective way of polysaccharide.
The most frequently used sulphating reagent is chlorsulfonic acid-pyridine complex, because its productive rate and substitution value are all ideal, is easy to obtain sulfated polysaccharides.
Document " Authorization Notice No. is CN101948549B Chinese invention patent " discloses a kind of sulfation gynostemma pentaphylla polysaccharide preparation method.The method adopts chlorsulfonic acid and pyridine to make sulfur acidizing reagent, respectively with temperature of reaction, in the reaction times, reactant ratio is as influence factor, adopt the method for Three factors-levels to obtain the sulphating gynostemma pentaphylla polysaccharide of 9 different degree of substitution, briefly understand that it is to the performance degree of immunity function.Whole process reaction transformation efficiency is higher, but has strong oxidizing property and pungency due to chlorsulfonic acid itself, and actual experiment operating process is comparatively loaded down with trivial details, and reaction risk level is high; Even more important some the method does not carry out analysis of immunogenicity experiment for different degree of substitution, namely final and relation between unresolved substitution value and immunocompetence.
Summary of the invention
In order to overcome the low deficiency of sulphating gynostemma pentaphylla polysaccharide immunocompetence prepared by existing method, the invention provides a kind of preparation method of sulphating gynostemma pentaphylla polysaccharide.The method adopts sulfur trioxide-pyridine as sulphating reagent, DMF is as reaction solvent, respectively with temperature of reaction, reaction times, reactant ratio is as influence factor, adopt the method for Three factors-levels to prepare gynostemma pentaphylla polysaccharide product, adopt bariumchloride-Turbidity of Gelatin method to survey its respective substitution value.Get mouse primary spleen lymphocyte and peritoneal macrophages respectively, employing mtt assay detection different degree of substitution; The impact of sulphating gynostemma pentaphylla polysaccharide product on cell proliferation.Simply controlled by present invention achieves preparation process, security is high, and can prepare the sulphating gynostemma pentaphylla polysaccharide product had compared with high immunological activity pointedly.
The technical solution adopted for the present invention to solve the technical problems is: a kind of preparation method of sulphating gynostemma pentaphylla polysaccharide, is characterized in adopting following steps:
Step one, in large container with the dry plant of 95% alcohol immersion gynostemma pentaphylla, remove ethanol afterwards, use 95% ethanol, 50 DEG C of refluxing extraction 2 times on a rotary evaporator, each 2h, extraction terminates rear filtration, volatilize ethanol, the dry thing obtained adds ultrapure water, 95 DEG C of refluxing extraction 3 times in large twoport flask, each 3-4 hour, filter with double gauze after end, collect filtrate, 50 DEG C of concentrating under reduced pressure, 20 DEG C, 7000rpm, after centrifugal 30min, gets supernatant liquor, add 4 times of volume 95% ethanol, 4 DEG C of precipitates overnight, collecting precipitation, centrifugal.Precipitation uses 95% ethanol, acetone, washed with diethylether, last lyophilize respectively, obtains gynostemma pentaphylla Crude polysaccharides powder.
Step 2, by the gynostemma pentaphylla Crude polysaccharides aqueous solution 30% strong aqua adjust ph to 8.0 of 0.5%, under agitation, add 30%H gradually in 50 DEG C
2o
2, be faint yellow to solution, constant temperature keeps 2h, uses CH
3cOOH adjust ph to 7.0, concentrating under reduced pressure.Add 95% ethanol of 4 times of supernatant volumes, 4 DEG C are spent the night, and 7000rpm, 15min are centrifugal, collecting precipitation, use 95% ethanol, acetone, washed with diethylether successively, lyophilize.Obtain refined polysaccharide, called after GPMP.
Step 3, use N, dinethylformamide DMF makes solvent, dissolve sulfur trioxide-pyridine mixture, afterwards refined polysaccharide is joined in complex systems, use solvent wash beaker, after stirring and dissolving, system is moved in the three-necked bottle with thermometer and whipping appts, Keep agitation, 60 DEG C of water-baths.Be cooled to room temperature after reaction terminates, adjust PH to 7.0 with NaOH, alcohol settling spends the night, and next day is centrifugal, distill water dialysis, lyophilize, obtains the sulphating gynostemma pentaphylla polysaccharide product of different degree of substitution, called after S1 successively, S2, S3.
The step of described dissolving sulfur trioxide-pyridine mixture: add according to the amount of 2.5g sulfur trioxide-pyridine/20mL DMF.
The step of described tune PH, uses the NaOH solution of 5%.
The step of described alcohol precipitation is: the ethanol adding total liquor capacity before 3 times, 4 DEG C of hold over night.
Described centrifugal step is: use high speed freezing centrifuge, 7000rpm, centrifugal 15min.
The step of described dialysis for: the dialysis tubing molecular weight cut-off used is 3500, distill water dialysis 120h.
When step 4, detection substitution value, the drafting of typical curve: take sodium sulfate as standard substance, arranging concentration is respectively 20,40,60,80,100 μ g/mL, 1mL, does two parallel group, adds containing 0.5% bariumchloride respectively, the BaCl of 0.5% gelatin
2-Gel solution, and gelatin solution, after mixing mutually, at room temperature place 15-20min with 3% trichoroacetic acid(TCA), is determined at the absorbancy at 360nm place, and with the quality of sulfur-bearing for X-coordinate, the difference A1-A2 of two groups of absorbancys is ordinate zou, obtains typical curve.
By sample dissolution in 1mol/L hydrochloric acid soln, 100 DEG C of hydrolysis 1h in test tube with ground stopper with a scale.Filter after hydrolysis completely, measure A1-A2 by typical curve working method, calculate the content S of sulphur according to regression curve.
The calculation formula of sample substitution value: DS=1.62 × S%/(32-1.02 × S%)
In formula: DS represents substitution value, S% represents sulphur content.
The invention has the beneficial effects as follows: adopt sulfur trioxide-pyridine as sulphating reagent, DMF is as reaction solvent, respectively with temperature of reaction, reaction times, reactant ratio is as influence factor, adopt the method for Three factors-levels to prepare gynostemma pentaphylla polysaccharide product, adopt bariumchloride-Turbidity of Gelatin method to survey its respective substitution value.Get mouse primary spleen lymphocyte and peritoneal macrophages respectively, employing mtt assay detection different degree of substitution; The impact of sulphating gynostemma pentaphylla polysaccharide product on cell proliferation.Simply controlled by present invention achieves preparation process, security is high, and can prepare the sulphating gynostemma pentaphylla polysaccharide product had compared with high immunological activity pointedly.
1. the present invention adopts the method Gynostemma pentaphyllum Makino polysaccharide of sulfur trioxide-pyridine to carry out sulphating modification, and reaction process security is high, is easy to control and operation.
2. adopt sulfur trioxide-pyridine mixture 2.5g, temperature of reaction 60 DEG C, reaction times 2h, can obtain the product that substitution value is 0.253, called after S1; Sulfur trioxide-pyridine mixture 5g, temperature of reaction 60 DEG C, reaction times 4h, obtains the product that substitution value is 0.76, called after S2; Sulfur trioxide-pyridine mixture 5g, temperature of reaction 60 DEG C, reaction times 6h, obtains the product that substitution value is 0.82, called after S3.
3. the action effect of sulphating gynostemma pentaphylla polysaccharide product S1 increases with the increase of concentration, and when concentration is 200 μ g/mL, effect reaches best, and when concentration is greater than 200 μ g/mL, activity is successively decreased; Product S2 action effect increases with the increase of concentration, reaches best to effect during 800 μ g/mL; The action effect of product S6 increases with the increase of concentration, and when concentration is 100 μ g/mL, effect reaches best.
4. sulphating gynostemma pentaphylla polysaccharide product collaborative ConA promotes the proliferation rate of mouse spleen lymphocyte: S1 is 26.26%, S2 be 142.52%, S3 is 9.08%; Sulphating gynostemma pentaphylla polysaccharide product promotes the proliferation rate of mouse peritoneal macrophages: S1 is 23.64%; Sample S2 promotes that the proliferation rate of mouse peritoneal macrophages is 69.70%; Product S3 promotes that the proliferation rate of mouse peritoneal macrophages is 38.18%..
The pass obtained between sulphating gynostemma pentaphylla polysaccharide product substitution value and immunocompetence is: the polysaccharide product after sulphation modification, its immunocompetence all has raising in various degree, wherein substitution value be 0.76 product S2 immunocompetence be significantly higher than the sulfation gynostemma pentaphylla polysaccharide product of other substitution values.
Below in conjunction with the drawings and specific embodiments, the present invention is elaborated.
Accompanying drawing explanation
Fig. 1 is the impact that sulphating gynostemma pentaphylla polysaccharide product S1, S2, S3 of different degree of substitution works in coordination with LPS and breed mouse spleen lymphocyte.
Fig. 2 is the impact that sulphating gynostemma pentaphylla polysaccharide product S1, S2, S3 of different degree of substitution breeds Turnover of Mouse Peritoneal Macrophages.
Embodiment
Following examples are with reference to Fig. 1-2.
The extraction of embodiment one, gynostemma pentaphylla polysaccharide.
Take the dry plant 500g of gynostemma pentaphylla (being purchased from Xi'an Chinese Medicinal Materials Markets), with 95% alcohol immersion 48h in large container, remove ethanol afterwards, use 95% ethanol, 50 DEG C of refluxing extraction 2 times on a rotary evaporator, each 2h, extraction terminates rear filtration, volatilize ethanol, the dry thing obtained adds ultrapure water, 95 DEG C of refluxing extraction 3 times in large twoport flask, each 3-4 hour, filter with double gauze after end, collect filtrate, 50 DEG C of concentrating under reduced pressure, 20 DEG C, 7000rpm, after centrifugal 30min, get supernatant liquor, add 4 times of volume 95% ethanol, 4 DEG C of precipitates overnight, collecting precipitation, centrifugal.Precipitation uses 95% ethanol, acetone, washed with diethylether, last lyophilize respectively, obtains gynostemma pentaphylla Crude polysaccharides powder.
Embodiment two, gynostemma pentaphylla Crude polysaccharides depigmentation.
By 0.5% Crude polysaccharides aqueous solution strong aqua adjust ph to 8.0, under agitation, add 30%H gradually in 50 DEG C
2o
2, be faint yellow to solution, constant temperature keeps 2h, uses CH
3cOOH adjust ph to 7.0, concentrating under reduced pressure.Add 95% ethanol of 4 times of supernatant volumes, 4 DEG C are spent the night, centrifugal (7000rpm, 15min), and collecting precipitation uses dehydrated alcohol, acetone, washed with diethylether, lyophilize successively.Obtain refined polysaccharide, called after GPMP.
The sulphation modification of embodiment three, gynostemma pentaphylla polysaccharide.
This law adopts the method Gynostemma pentaphyllum Makino polysaccharide of sulfur trioxide-pyridine to carry out sulphating modification.Determine that different reaction times, differential responses thing ratio are on the impact of sulphating gynostemma pentaphylla polysaccharide reaction process respectively, substitution value is index.
In beaker, a certain amount of sulfur trioxide-pyridine mixture DMF is dissolved, take 500mg gynostemma pentaphylla polysaccharide afterwards, join in system, after dissolving to be mixed, system is moved in the there-necked flask with thermometer and whipping appts, 60 DEG C of water-bath certain hours.Be cooled to room temperature after reaction terminates, with 5%NaOH, system PH be adjusted to 7.0, after add 95% ethanol of 3 times of systems, 4 DEG C of hold over night.Next day until precipitation precipitation after, 7000rpm, centrifugal 15min, collecting precipitation.Precipitation is dissolved in distilled water, use molecular weight cut-off be the dialysis tubing of 3500, distill water dialysis 120h, postlyophilization obtain sulfation gynostemma pentaphylla polysaccharide product.
The substitution value of embodiment four, sulfation gynostemma pentaphylla polysaccharide measures.
The drafting of typical curve: take sodium sulfate as standard substance (20,40,60,80,100 μ g/mL, 1mL), do two parallel group, add 1%BaCl respectively
2-Gel solution (0.5% bariumchloride, 0.5% gelatin) 0.625mL, and gelatin solution, after mixing mutually with 3% trichoroacetic acid(TCA), at room temperature place 15-20min, be determined at the absorbancy at 360nm place, with the quality of sulfur-bearing for X-coordinate, the difference (A1-A2) of two groups of absorbancys is ordinate zou, obtains typical curve.
Get 10mg sample and in test tube with ground stopper with a scale, be dissolved in 1mL 1mol/L hydrochloric acid at 100 DEG C of hydrolysis 1h.Filter after hydrolysis completely, get 0.2mL and measure (A1-A2) by typical curve working method, calculate the content S of sulphur according to regression curve.
The calculation formula of sample substitution value: DS=1.62 × S%/(32-1.02 × S%)
In formula: DS represents substitution value, S% represents sulphur content.
The sulphating gynostemma pentaphylla polysaccharide of the different degree of substitution obtained under table 1 differential responses condition
Mass of GPMP was maintained in 500mg,the solvent was DMF,temperature was maintained 60℃
With reference to table 1, record the substitution value of three different sulphating gynostemma pentaphylla polysaccharide products, S1 measurement result is 0.253, S2 be 0.76, S3 is 0.82.
The impact that embodiment five, sulphating gynostemma pentaphylla polysaccharide are bred mouse spleen lymphocyte.
Get the BALB/c mouse in 18-22g 6-8 age in week, de-neck is put to death, small pieces are cut into after taking out spleen, grind (200 order) on the net at stainless steel sift and obtain splenocyte suspension, after adding the standing 4-5min of erythrocyte cracked liquid Tris-HCl-NH4Cl (pH 7.2) mixing in suspension, 2000rpm is centrifugal, abandon supernatant, being mixed with concentration is 2 × 10
6the spleen lymphocyte suspension of individual/mL.Cell suspension is added in 96 orifice plates, every hole 100 μ L, after inserting incubator cultivation 4-6h, add different concns (0,25,50,100,200,400,800 μ g/mL) and ConA (5 μ g/mL)/LPS (the 10 μ g/mL) sample of GPMP, S1, S2, S6 again, make the independent or collaborative ConA/LPS of polysaccharide sample act on spleen lymphocyte, in negative control, only add nutrient solution and ConA/LPS.System is placed in 37 DEG C, the CO of 5%
2after hatching 72h in incubator, every hole adds 20 μ L CCK-8 solution, after continuing to hatch 4h, microplate reader detects the absorbance under 450nm condition.
Table 2 different concns gynostemma pentaphylla polysaccharide and sulphating derivative product collaborative ConA thereof are to the Effect of promoting growth of mouse lymphocyte
*P＜0.05,
*P＜0.01
***P＜0.001vs ConA Group；
#P＜0.05,
##P＜0.01,
###P＜0.001vs GPMP Group,
&&&P<0.001vs Control Group
With reference to table 2, sample S1 works in coordination with ConA and acts on mouse spleen lymphocyte, and in the scope of 25-100 μ g/mL concentration, along with the increase of concentration, appreciation rate increases, and when concentration is 100 μ g/mL, appreciation rate is maximum, increases appreciation rate afterwards reduce successively with concentration; Sample S2 works in coordination with ConA in the scope of 25-100 μ g/mL concentration, and along with the increase of concentration, lymphocyte proliferation rate increases; Sample S6 is in the scope of 25-50 μ g/mL concentration, and along with the increase of concentration, appreciation rate increases, and when concentration is 50 μ g/mL, appreciation rate is maximum, increases appreciation rate afterwards reduce successively with concentration.With reference to Fig. 1, it be 26.26%, S2 be 142.52%, S3 to mouse spleen lymphocyte appreciation rate: S1 is 9.08% that sulphating gynostemma pentaphylla polysaccharide S1, S2, S3 work in coordination with ConA.
The impact that embodiment six, sulphating gynostemma pentaphylla polysaccharide are bred Turnover of Mouse Peritoneal Macrophages.
De-neck puts to death mouse, and get peritoneal macrophage, being made into concentration is 6 × 10
5the cell suspension of individual/mL.Cell suspension is added in 96 orifice plates, every hole adds 100 μ L, after inserting incubator cultivation 4-6h, add different concns (0,25,50,100,200,400, the 800 μ g/mL) sample of GPMP, S1, S2, S6 again, only add nutrient solution in negative control, system is placed in 37 DEG C, the CO of 5%
2after hatching 72h in incubator, every hole adds 20 μ L MTT, continues to hatch 4h, abandons supernatant afterwards, and every hole adds 150 μ L DMSO, under the condition of 570nm, measure its absorbance by microplate reader.
Table 3 different concns gynostemma pentaphylla polysaccharide and sulphating derivative product thereof promote to use to the propagation of Turnover of Mouse Peritoneal Macrophages
*P＜0.01,
***P＜0.001vs Control Group；
#P＜0.01,
###P＜0.001vs GPMP Group
With reference to table 3, sample S1 acts on mouse peritoneal macrophages, and in the scope of 25-100 μ g/mL concentration, along with the increase of concentration, appreciation rate increases, and when concentration is 100 μ g/mL, appreciation rate is maximum, increases appreciation rate afterwards reduce successively with concentration; Sample S2 is in the scope of 25-100 μ g/mL concentration, and along with the increase of concentration, scavenger cell appreciation rate increases; Sample S6 is in the scope of 25-50 μ g/mL concentration, and along with the increase of concentration, appreciation rate increases, and when concentration is 50 μ g/mL, appreciation rate is maximum, increases appreciation rate afterwards reduce successively with concentration.With reference to Fig. 2, sulphating gynostemma pentaphylla polysaccharide sample S1, S2, S3 promote the proliferation rate of mouse peritoneal macrophages: S1 is 23.64%; S2 is 69.70%; S3 is 38.18%.
Claims (1)
1. a preparation method for sulphating gynostemma pentaphylla polysaccharide, is characterized in that comprising the following steps:
Step one, in large container with the dry plant of 95% alcohol immersion gynostemma pentaphylla, remove ethanol afterwards, use 95% ethanol, 50 DEG C of refluxing extraction 2 times on a rotary evaporator, each 2h, extraction terminates rear filtration, volatilize ethanol, the dry thing obtained adds ultrapure water, 95 DEG C of refluxing extraction 3 times in large twoport flask, each 3-4 hour, filter with double gauze after end, collect filtrate, 50 DEG C of concentrating under reduced pressure, 20 DEG C, 7000rpm, after centrifugal 30min, gets supernatant liquor, add 4 times of volume 95% ethanol, 4 DEG C of precipitates overnight, collecting precipitation, centrifugal; Precipitation uses 95% ethanol, acetone, washed with diethylether, last lyophilize respectively, obtains gynostemma pentaphylla Crude polysaccharides powder;
Step 2, by the gynostemma pentaphylla Crude polysaccharides aqueous solution 30% strong aqua adjust ph to 8.0 of 0.5%, under agitation, add 30%H gradually in 50 DEG C
2o
2, be faint yellow to solution, constant temperature keeps 2h, uses CH
3cOOH adjust ph to 7.0, concentrating under reduced pressure; Add 95% ethanol of 4 times of supernatant volumes, 4 DEG C are spent the night, and 7000rpm, 15min are centrifugal, collecting precipitation, use 95% ethanol, acetone, washed with diethylether successively, lyophilize; Obtain refining gynostemma pentaphylla polysaccharide;
Step 3, make solvent with DMF, dissolve sulfur trioxide-pyridine mixture, add according to the amount of the DMF of 2.5g sulfur trioxide-pyridine/20mL; Afterwards refined polysaccharide is joined in complex systems, use solvent wash beaker, after stirring and dissolving, system is moved in the three-necked bottle with thermometer and whipping appts, Keep agitation, 60 DEG C of water-baths; Be cooled to room temperature after reaction terminates, adjust PH to 7.0 with 5%NaOH, ethanol 4 DEG C of precipitates overnight of 3 times of volumes, next day 7500rpm, 15min is centrifugal, distill water dialysis 120h, dialysis tubing molecular weight cut-off is 3500, lyophilize, obtains the sulphating gynostemma pentaphylla polysaccharide of different degree of substitution.
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| CN107625783A (en) * | 2017-08-09 | 2018-01-26 | 天津科技大学 | Sulfation chlorella, spirulina complex polysaccharide immunomodulating regimens application |
| CN116731221A (en) * | 2023-06-26 | 2023-09-12 | 程晓琳 | Polysaccharide derivative and application thereof in pharmacy |
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| CN116731221A (en) * | 2023-06-26 | 2023-09-12 | 程晓琳 | Polysaccharide derivative and application thereof in pharmacy |
| CN116731221B (en) * | 2023-06-26 | 2024-03-12 | 益得来(深圳)健康科技有限公司 | Polysaccharide derivative and application thereof in pharmacy |
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